Cellular access of iron (Fe) is limited to protect against pathological consequences of free radical damage and the precipitation of iron oxides. This is reflected in the cell's disposition to sequester Fe within specialized domains, like endocytic vesicles and mitochondria, and in proteins, like ferritin and transferrin (Tf). A key problem that remains to be solved is how Fe passes across membrane bilayers in a manner that responds to metabolic demands. Mediators that control transport are obscure, mechanisms of transport across membranes are poorly understood, and both issues are extremely important with respect to iron-deficient state of anemia and iron-overload states of hemochromatosis. Our long- term objective is to elucidate the mechanism through which Fe is translocated across membrane bilayers. We will focus on the two known routes of Fe delivery - an intracellular, Tf-dependent, transport system and a cell surface, non-endocytic, mechanism. Non-endocytic uptake of Fe is a multi-functional process of Fe3+-binding, reduction to Fe2+, and transport of Fe2+ across the bilayer. A novel integral membrane Fe3+- binding protein has been identified in rat liver plasma membranes that may function in the cell surface transport system. A specific goal of this project is to purify the Fe-binding protein and to investigate its potential role in transport using a newly-developed ferritin/ferrozine- coupled reconstitution system. with this unique model system, other transport factors and/or accessory proteins that function in the translocation of Fe across lipid bilayers can be identified. A second goal is to monitor the efflux of Fe from intracellular compartments containing endocytosed FeTf. Previous work has established a cell-free system that reconstitutes elements of receptor-mediated endocytosis of Tf by K562 cells. Using a modification of this system, the transport of radiolabel from the lumen of endocytic vesicles loaded with 55FeTf will be reconstituted. Analysis of Fe efflux in the cell-free assay will identify the parameters necessary for endosomal transport, including potential requirements for cytosolic factors. These studies will also identify which endocytic organelle houses the transmembrane translocation apparatus (early endosome, late endosome, or lysosome). It is possible that cell surface transporters are the same as/or different from intracellular transporters. As a final goal, characteristics of cell surface and intracellular transport mechanisms will be examined in order to identify potential relationships. Substantial characterization of cell surface Fe transport by K562 cells has already been completed. Using the cell-free assay, the properties of the intracellular transmembrane transport will be defined. This study will compare Km and Vmax values, cation specificity and sensitivity to inhibitors. Not only will these functional properties be examined, but regulatory aspects will also be explored to determine how cell surface and intracellular transport mechanisms may differ. These combined efforts will define whether plasma membrane and endosomal transporters share common elements or if unique uptake systems exist to translocate Fe across specific membrane bilayers.